Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Repeated siRNA application is a precondition for successful mRNA gammaENaC knockdown in the murine airways.

The volume of the airway surface liquid is regulated by Na(+) absorption and Cl(-) secretion by the respiratory epithelium. In cystic fibrosis, Na(+) hyperabsorption caused by the absence of functional CFTR protein leads to an altered airway surface liquid composition and finally to a deteriorated mucociliary clearance. It has been suggested that down regulation or inhibition of the amiloride-sensitive epithelial Na(+) channel (ENaC) could restore the disrupted airway hydration. Therefore, targeting ENaC by RNA interference could be of therapeutic relevance. In this context, we investigated whether RNAi could lead to a reduction in gammaENaC expression in epithelia in vitro and in vivo in mice. Transfection of cells with specific siRNA sequences for gammaENaC subunit reduced expression to approximately 10% relative to control. For in vivo experiments, siRNA sequences specific for the gammaENaC subunit were administered to the murine nasal cavity and, 72h later the animals were killed. In the first approach, only a single application of naked siRNA was given. In the second approach, repeated siRNA applications were performed. The single application of siRNA sequences had no effect on mRNA content of the targeted gammaENaC subunit, whereas repeated siRNA application resulted in a significant reduction in gammaENaC mRNA in the respiratory tissue. We conclude that repeated siRNA application is necessary for gammaENaC knockdown in the murine airways.